Osteocrin (OSTN) is a secreted protein predominantly expressed in bone and muscle tissues. As an endogenous ligand for natriuretic peptide receptors, it plays a crucial role in regulating bone growth, metabolism, and musculoskeletal development. Structurally, OSTN shares similarities with natriuretic peptides and modulates cardiovascular and skeletal homeostasis through competitive binding. Notably, it binds with high affinity to Natriuretic Peptide Receptor 3 (NPR3). This binding interaction inhibits the degradation of other natriuretic peptides, thereby enhancing their bioavailability and prolonging their physiological effects on blood pressure regulation and cardiovascular protection.To detect the activity of recombinant OSTN a functional ELISA assay was performed to evaluate the interaction between recombinant rat OSTN and recombinant rat NPR3.Briefly, OSTN was diluted serially in PBS with 0.01% BSA (pH 7.4). Duplicate samples of 100 μl were then transferred to NPR3-coated microtiter wells and incubated for 1h at 37℃. Wells were washed with PBST and incubated for 1h with anti-OSTN pAb, then aspirated and washed 3 times. After incubation with HRP labelled secondary antibody for 1h at 37℃, wells were aspirated and washed 5 times. With the addition of substrate solution, wells were incubated 15-25 minutes at 37℃. Finally, add 50 μL stop solution to the wells and read at 450/630nm immediately. The binding activity of recombinant rat OSTN and recombinant rat NPR3 was shown in Figure 1, the EC50?for this effect is 0.10 μg/mL.